Papillomaviruses induce benign, dysplastic and malignant hyperproliferations of skin or mucosal epithelium. Pfister, Rev. Physiol. Biochem. Pharmacol., 99:111-181 (1984). According to Nuovo et al., J. Virol., 62:1452-1455 (1988) 51 types (strains) of human papillomavirus (HPV) have been identified.
In humans, different papillomavirus types are known to cause distinct diseases, Pfister, Adv. Cancer Res., 48:113-147 (1987), Syrjanen, Obstet. Gynecol. Survey, 39:252-265 (1984). For example, human papillomavirus (HPV) types 1 and 2 cause common warts, and types 6 and 11 cause condylomas and genital flat warts. In contrast, HPV types 16, 18 and 33 are carried in a majority of cervical cancers and do not cause the usual condyloma but rather persist diffusely in the cervical endothelium exhibiting only minimal pathologic changes. It is believed that the HPV types associated with cervical cancer are maintained in a latent state in cervical endothelium tissues for years after initial infection and then progress in some cases to cause cervical cancer.
The genome of many of the presently identified HPV types has been cloned and sequenced. See, for example, Baker, "Sequence Analysis of Papillomavirus Genomes", in The Papovaviridae-Volume 2: The Papillomaviruses, Salzman et al., eds., Plenum Press, New York, pp. 321-386 (1987); and Chow et al., Cancer Cells, 5:55-72 (1987).
Historically, the open reading frames (ORFs) of papillomavirus genomes have been designated L1 and L2 and E1 to E7, where "L" and "E" denote late and early, respectively. L1, L2 and E4 code for viral capsid proteins and E region ORFs are thought to be associated with functions such as viral replication, transformation and plasmid maintenance. Howley et al , "Molecular Aspects of Papillomavirus-Host Cell Interactions" in Viral Etiology of Cervical Cancer, Peto et al., eds., Banrury Report 21, Cold Spring Harbor Laboratory, pp. 261-272 (1986); and Doorbar et al., EMBO J., 5:355-362 (1986).
Presently, there are no papillomavirus-specific antigens that have been unambiguously identified as being either expressed during, or indicative of, latent HPV infection.
This is in contrast to HPV infected tissues where there are actively replicating viruses. In those tissues the presence of some HPV-encoded replication-related antigens (e.g., viral capsid antigen) has been demonstrated. Schneider, "Methods of Identification of Human Papillomaviruses," in Papillomaviruses and Human Disease, Syrjanen et al., eds., Springer-Verlag, pp. 19-39 (1987).
Several studies have reported attempts to identify the protein products of HPV-containing cell lines. Fusion proteins were expressed in Escherichia coli in which various HPV ORF region nucleotide sequences were operatively linked to heterologous genes. The resulting fusion protein product contained a non-HPV amino terminus and part or all of the putative ORF-encoded amino acid residues at the carboxy terminus. The expressed fusion protein was used as an immunogen to raise polyclonal antisera, and the sera was then used to detect putative HPV-encoded proteins in vitro in HPV-containing cell lines.
For instance, Seedorf et al., EMBO J., 6:139-144 (1987) raised antibodies to a fusion protein containing E1 ORF sequences and detected a 70 kilodalton (kd) protein after in vitro translation of mRNA isolated from HeLa cells containing HPV type 18. Using antisera raised against a fusion protein containing E4 ORF sequences, a 10 kd protein was detected by in vitro translation of MRNA from HPV type 16-containing CaSki cells. Seedorf et al., EMBO J., 6:139-144 (1987). Similarly, antisera directed against an E6 ORF sequence-derived fusion protein detected an 11 kd protein by in vitro translation of mRNA from HPV type 16-containing CaSki cells. Seedorf et al., EMBO J., 6:139-144 (1987).
Antisera raised to various fusion proteins that contained E7 ORF sequences have detected several proteins depending on the HPV type studied. In HPV 16 infected cells, a 15 kd protein has been detected using Western immunoblotting and radioimmuno-precipitation methodologies using CaSki or SiHa cells as the HPV source. Seedorf et al., EMBO J., 6:139-144 (1987); and Firzlaff et al., Cancer Cells, 5:105-113 (1987). Smotkin et al., Proc. Natl. Acad. Sci. USA, 83:4680-4684 (1987) have described using antibodies raised against an E7 ORF sequence-derived fusion protein to detect a 20 kd protein by immunoprecipitation of HPV type 16-containing CaSki or SiHa cells.
Monoclonal antibodies have been prepared against an E7 ORF-containing fusion protein that detect a 15 kd protein in HPV 16-containing cells by using both Western and immunoprecipitation methodologies. Oltersdorf et al., J. Gen. Virol., 68:2933-2938 (1987).
Recently, Li et al., (J. Gen Virol., 62:606-609 (1988)) described an antisera raised against an E2 ORF containing fusion protein which was used to detect proteins present in primary biopsy tissues known to contain HPV genomic sequences. A 50 kd protein was detected by Western immunoblotting of lysates from several tissues diagnosed as condylomas and demonstrated by Southern blotting to contain HPV types 6, 11 or 16.
By way of further background, seventeen synthetic polypeptides have been described whose amino acid residue sequences correspond to portions of the HPV type 16 E1, E2, E4, E6, or E7 ORFs, or to a portion of the E6 ORF region of HPV type 6. Schoolnik et al., EPO patent application no. 0257754A2, published Mar. 2, 1988. These polypeptides were used as immunogens to prepare rabbit antisera, and four of the prepared anti-peptide antibodies raised against an E6 region of HPV-16 were shown to immunoreact with patient biopsy tissue shown to contain HPV-16 DNA and that were assessed as having known dysplasias. However, none of the Schoolnik et al. peptides was demonstrated as having the ability to react as an antigen with antibodies induced as a result of HPV infection.